Growing interest in immunohistochemical staining procedures has led to the development of a wide range of highly specific immunostains which are of value to the surgical pathologist in diagnostic and investigative studies (1, 2). Although formalin remains the most popular fixative used in pathology, it is clear that this fixative is not always the best choice for preserving antigenicity of tissues to be used in immunohistochemical procedures Despite numerous studies on the intermolecular crosslinks formed between formalin and proteins (3, 4), the molecular mechanism underlying tissue fixation is not well understood (5).
The demand for a broader selection of antibodies which can be used for immunohistochemical staining on routine formalin-fixed, paraffin-embedded tissues has stimulated efforts to develop antibodies which can recognize formalin-resistant epitopes Although this strategy has been effective in developing many useful antibodies, it has not been entirely satisfactory in resolving all problems. A persistent concern in immunopathology is choosing the correct fixative and duration of fixation that will provide the maximum preservation of tissue morphology with minimum loss of antigenicity.
One approach to resolve this dilemma was the introduction of protease digestion of formalin-fixed sections to unmask antigenic sites hidden by cross-linked proteins (6, 7). However, Leong, et al , (8) showed that, aside from cytokeratins and desmin, digestion with trypsin did not improve immunostaining of the other antigens studied. Presently it is not clear whether or not the formalin-induced cross-linking of proteins is a reversible chemical reaction. However, a recent study concerning formalin sensitivity of a GFAP epitope supported the hypothesis that the sensitivity of some epitopes was not due to the direct effect of the aldehyde, but rather due to the binding of other molecular structures to the epitope (9).
Clearly the ability to bind immunostaining reagents with epitopes masked by formalin fixation (referred to here as antigen-retrieval) could significantly expand the range of antibodies useful in immunohistochemistry as well as reduce the incidence of false negative staining in over-fixed tissues. Additionally, antigen retrieval could provide greater diagnostic accuracy by improving immunohistochemical procedures. With these goals in mind we studied the effects of microwave oven heating of tissue sections in the presence and absence of metal solutions. The dramatic enhancing effect of this treatment on antigen recovery and immunohistochemical staining was particularly surprising considering the deleterious effects that high temperatures are presumed to have on protein antigens.
The microwave oven has previously been used for tissue fixation (8, 10, 11) and for rapid histochemical and immunohistochemical staining (12-19). One recent report has also observed enhanced immunohistochemical staining following microwave drying of slides (20). However, in these cases, only short periods of irradiation and low temperatures were used To the best of our knowledge, no one has previously accomplished antigen retrieval by the use of the microwave oven alone or with metal solutions. Similarly, no studies have indicated that immunohistochemical staining intensity could be increased by heating slides in water with microwaves to temperatures of approximately 100.degree. C.
The use of heavy metal salts in combination with formalin for tissue fixation has recently been introduced (21, 22). Some studies have demonstrated the superiority of zinc formalin as a fixative for antigen preservation (22) Furthermore, when routine formalinfixed tissues were re-fixed in zinc formalin, immunoreactivity was improved (23).
Although little has been published on the molecular changes in amino acids and other compounds that occur after microwave treatment (24), Stroop, et al., (25) demonstrated that microwave treatment of radiolabeled DNA probes allowed these probes to be diluted about 20 times more than when these probes were denatured by conventional heat.